The inactive state of the small G Protein Cdc42, the Cdc42 . GDP . Mg2+ ternary complex, was investigated using fluorescence, Mn2+ substituted electron paramagnetic resonance, and P-31 nuclear magnetic resonance spectroscopy at various urea concentrations. The urea interaction with the protein was used to probe the binding state of GDP . Mg2+ to Cdc42. Two binding states of the Cdc42 . GDP . Mg2+ ternary complex with different binding stability were observed. The two binding states were characterized by two sets of P-31 resonance of GDP phosphate groups, namely P-alpha and P-beta, P'(alpha), and P'(beta). The high populated binding state I (P-alpha and P-beta) was more stable and less sensitive to the urea interaction. Yet the population of binding state II (P'(alpha) and P'(beta)) was lower, and the binding of GDP . Mg2+ to Cdc42 in this state was more sensitive to the urea interaction. The release of GDP . Mg2+ from the ternary complex in binding state II was faster than in state (C) 2002 Elsevier Science (USA).